We propose to develop an approach for the identification of cis-regulatory sequence elements in mammalian genomes. Our approach calls for simultaneous expression profiling and TF-binding-location analysis under multiple time points and conditions relevant to a specific biological process of interest. The TF-binding regions for co-regulated genes, and their orthologous sequences in multiple mammalian species will then be analyzed in order to extract the cis-regulatory elements that mediate the co-regulation. The primary goals are to develop the computational algorithms and software that are necessary for the extraction of cis-regulatory modules based on the experimental data, and to apply this approach to the study of Sonic Hedgehog (Shh) responsive gene regulation in mouse embryonic development. The significance of this work stems from the fact that these cis-regulatory sequence elements are a key part of the "control signals" in the genome that direct the temporally and spatially specific transcription of genes. By developing the general computational tools for identifying these elements, this project will contribute significantly to the understanding of the functions of the genome. This project will also generate direct knowledge on cis-elements responsive to Shh signaling, which is fundamental in numerous processes in developmental and cancer biology. Our specific aims are: 1) Development of statistical methods and computational algorithms for identifying cis-regulatory modules. 2) Development of a software application to support studies of gene regulation from combined expression profiling and TF-binding location data. 3) Identification of cis-regulatory elements controlling Shh- responsive transcriptional events in selected developmental processes in mouse using the proposed approach.